Human Y-interferon signal peptide-tumor necrosis factor(TNF) gene fusions

ABSTRACT

A virion expression system for a desired protein packaged in an envelope derived from a retrovirus useful in administering proteins which cross cell membranes in order to serve their function. Preferred virions are those that carry an RNA sequence that encodes cytokines or lymphokines, and includes IL-2, multiple drug resistance protein, and TNF. Particularly disclosed is a DNA construct in which a gene encoding tumor necrosis factor (TNF) is directly linked to DNA encoding a human γ-interferon signal peptide.

This application is a continuation of U.S. Ser. No. 07/488,706, filedMar. 2, 1990 now abandoned which in turn is a continuation-in-part ofU.S. Ser. No. 07/474,169, filed on Feb. 2, 1990, now abandoned which inturn is a continuation-in-part of U.S. Ser. No. 07/426,986, filed onOct. 24, 1989 now abandoned.

TECHNICAL FIELD

The invention relates to the use of DNA recombinant technology to effectprotein delivery to cells. In particular, it concerns the use of highand low titer recombinant retroviral vectors to deliver one or moredesired protein(s) to cells, or a host organism that would benefit fromthe presence of the protein. A wide variety of proteins can be deliveredincluding tumor necrosis factor, Interleukins, in particular InterleukinII, and the protein that confers multiple drug resistance on cells.

BACKGROUND ART

So many approaches have been used to effect delivery of proteins,particularly proteins that have medically beneficial applications, todesired target cells that a survey of this field would be bothinappropriate and unhelpful. It should be noted, however, that virtuallyall delivery systems presently employed address the problem ofpenetrating barriers to the circulatory system of the subject organismand do not address the problem of uptake by particular cells targetedfor treatment with the protein. Thus, in the simplest form of ensuringpenetration of these barriers, intravenous injection of a solution of anactive ingredient, delivery of the protein merely results in the activeingredient circulating in the blood, but without provision for anyspecial mechanism to ensure that the protein will find its way into thecytoplasm or nucleus of a cell that it is expected either to treat or tokill. While a specific cell may be targeted, e.g., through the use ofantibody, penetration through cellular membranes is effected by whatevermechanism(s) is normally used by cells, or the appropriate site oftreatment is necessarily extracellular.

It is, of course, established that viral particles are capable ofintroducing foreign nucleic acids and proteins into cells in the normalcourse of infection. Use of viral particles to transport geneticmaterial into target mammalian cells for purposes of gene therapyappears to be the major approach now being followed to develop thistechnique. See, e.g., McCormick, D., Bio/Technology (1985) 3: 689-693.In addition, Lang, R. A., et al., Cell (1985) 43: 531-542 were able touse a similar system with GM-CSF to induce autocrine growth in a murineblood-cell line. In the Lang work, a cDNA-encoding GM-CSF was insertedinto a Moloney murine leukemia-based vector under control of thepromoter/enhancer of the viral long-terminal repeat, and infectious,helper-free virus was produced by transfecting into the Ψpsi-2-packaging cell line. The GMV virus produced was able to effectGM-CSF production in a hemopoietic cell line. This ability has notheretofore been used to transport designated protein drugs in an intactorganism, however.

Retroviruses in particular have been used as vectors for foreign geneinsertion, and the biology of retroviruses is, to a significant degree,understood: Retroviruses consist of a single stranded RNA genomeencapsulated in a protein envelope. The genome itself, reading from the5' to 3' end, contains a cap, 5' untranslated region, a segment of RNAdesignated "Ψ" which is necessary for the RNA to be packaged intoprotein -i.e., a packaging site, and then the coding sequences forseveral proteins -the retroviral core protein (gag); reversetranscriptase, to facilitate an intermediate stage consisting of a DNAtranscript (pol) and the viral envelope or capsid protein (env), allfollowed by some 3' untranslated sequences. The three viral proteins areneeded for the infectivity of the viral genome; the packaging site isneeded to produce additional infective virus.

Retroviruses experience a "proviral" stage which contains adouble-stranded cDNA copy of the protein-encoding region of the RNA.However, in this stage, the untranslated 3' and 5' regions are modifiedto obtain, at either end of this protein-encoding cDNA, a long terminalrepeat (LTR) which provides the appropriate promoter and enhancersequences to effect DNA transcription as well astranscription-terminating sequences at operable positions with respectto the coding portions.

In ordinary infection, the proviral double-stranded cDNA can beintegrated into the host cell genome and from there effect theproduction of additional virus particles containing the RNA genomepackaged in its protein capsule. For this procedure to take place, it iscritical that the Ψ packaging site be present in the provirus.

It has occurred to others that the protein encoding sequences of theretroviruses could be replaced with those for a desired protein so as toemploy the expression systems of the virus when the modified virusinfects host cells. See, e.g., U.S. Pat. No. 4,405,712 and Lang (supra).However, in order to achieve this, the modified viral genome requires ahelper virus capable of synthesizing the capsid proteins and packagingthe RNA transcripts of the foreign DNA.

Thus, for "gene therapy" the proviral DNA form is inserted into asuitable vector, replicated and packaged into viral envelopes with theaid of a helper virus. For a general review, see Anderson, W. F.,Science (1984) 226: 401-409; Coffin, J., "Genome Structure", in RNATumor Viruses, Vol 2, Weiss et al., eds, 2ed, (1985), Cold SpringHarbor, N.Y.

The most commonly used retroviruses for study of gene therapy have beeneither the murine sarcoma virus (MSV) or the Moloney murine leukemiavirus (MoMLV). (Mann, R., et al., Cell (1983) 33: 153-159.) The proviralform of these retroviruses is isolated and inserted into more or lessstandard bacterial cloning vectors for amplification. The proviralinsert, which contains the gag-, pol and env-encoding mRNA flanked bylong terminal repeats containing the control sequences, along with apackaging site is then manipulated to replace the region containing theprotein-encoding RNA with the desired foreign gene. If this DNA istransfected into host cells which have been infected with complete virusor with defective virus lacking only the packaging site, the RNA whichis synthesized from the modified provirus is then packaged into virionsfor reinfection of another cell. This provides a mechanism forintroduction of the DNA encoding the desired active ingredient or druginto the cell by infection.

There are two ways to go about this. In one approach, the modifiedproviral DNA is transfected into cells which bear an infection from theunmodified virus, co-residing in the cell. The normal viral vectors willsynthesize the packaging materials and some of the mRNA produced by themodified provirus will be packaged in a manner analogous to the normalviral RNA and then can be used to infect target cell for the productionof protein. Along with these commandeered viral envelopes, however, willbe a certain number of repackaged normal viral RNAs which, if notseparated from the "delivery truck" viruses simply cause additionalvirus infection in host cells infected with the products of this virionproduction round.

In a more useful approach, the provirus cloning vector containing thedesired gene is used to transfect a cell which has been geneticallymodified to produce defective viral envelopes which contain no viralgenomic RNA-in-effect, empty delivery trucks. These cells are obtainedby integration of the proviral form of a mutant retrovirus lacking the Ψpackaging site, and several such cell lines are available in the art toall that request them. Two of these lines, designated Ψ-1 or Ψ-2 areextensively described in Mann, R., et al., Cell (1983) 33: 153-159(supra) and are made by transfecting host NIH 3T3 fibroblast cells witha plasmid containing MoMLV proviral inserts from which the Ψ packagingsite had been deleted. The Ψ-2 cells apparently produce several emptyviral envelopes per cell corresponding to the viral envelope of thenative virus in the course of a generation. When these cells aretransfected with proviral DNA containing both a foreign gene and thepackaging site, Ψ, they package the mRNA transcript from the proviralDNA containing the foreign gene into these empty envelopes to generatemodified viruses which can infect any cells (murine in this case) whichare normally hosts for MoMLV. It should be noted, however, that thisrecombinant, modified virus is defective in that it cannot cause theproduction of additional modified (or other) virions in the cell it"infects". It is able to cause the production of the protein the geneencodes in the "infected" cell, but the infection cannot spread toadditional cells because no additional virions are produced.

More useful than Ψ-2 for the preparation of medicaments in the presentinvention are the Ψ-AM lines, which are available from Cone, R. D., etal., Proc Natl Acad Sci (USA) (1984) 81: 6349-6353. These lines are alsoobtained by transfecting NIH 3T3 cells, but with a vector designatedpMAV-Ψ-. This vector also contains an insert of a defective proviruswhich lacks the Ψ packaging site. However, pMAV-Ψ- is a hybrid encodingthe gag-pol sequences of MoMLV and envelope sequences derived from theamphitropic virus 4070A. The empty capsids produced by these cell linespackage RNA transcripts of cotransfected modified proviral DNA toproduce pseudo viruses which recognize and infect human, rat, and mousecells.

It has recently been observed that retroviral vectors carrying gagsequences exhibit higher titers than viruses that lack these sequences.Bender, et al., 1987, J. of Virology, 61(5): 1639-1646. Such high titerviruses facilitate efficient infection of various cells/tissues that aretargets for gene therapy. It is thought that the high titers of theseviruses is related to the presence of gag region sequences thathithertofore were not thought to be involved in packaging of viral RNAinto virions, and thus may allow for more efficient packaging.Regardless, such high titer retroviral vectors will have applications ingene therapy.

Thus, the art provides a system for moving genes into susceptible cellswhich has been, in the past, employed only for gene therapy or forgeneration of autocrine growth factors. These methods inevitably utilizean ex-vivo exposure of targeted cells to the retroviral vector; forexample, in gene therapy, bone marrow cells are removed and treated, andthen reimplanted. In the present invention, an analogous system ismustered to deliver pharmaceuticals to target cells using conventionalmethods of administration to produce a highly dead-end, localized"infection".

Disclosure of the Invention

The invention is directed to highly unusual pharmaceutical compositionsand to methods for delivering active drugs to cells of organismssusceptible to viral infection. The target organisms are ordinarilyvertebrates. In one embodiment, the pharmaceutical composition iscomposed of delivery viruses which contain envelope proteins capable ofcausing transient and nonreplicative infection of the cells in thesubject organism to be treated with the drug. These pharmaceuticalcompositions are administered by injection into the blood stream or bylocalized injection into masses of undesirable cells such as tumorcells. Alternatively, cells susceptible to viral infection may beremoved from the host organism infected with the appropriate virus andthen returned to the host organism where they secrete the desiredprotein drug.

Thus, in one aspect, the invention relates to a drug delivery systemwhich comprises a delivery retrovirus. The retrovirus has a "genome"comprising an RNA which encodes the desired active protein ingredientoperably linked to control sequences which were derived from aretrovirus and to a Ψ packaging site, and an envelope protein which iscapable of effecting the infection of a target host cell with thevirion, so that the target host cell alone is "infected", but unable topass this infection to additional cells.

In a second aspect of the invention, a high titer retroviral drugdelivery system is described wherein the high titer characteristics ofthe system are derived from the presence of gag sequences present in thevector. This vector is particularly useful for transforming cells ortissues that require high titers of virus, preferably tumor infiltratinglymphocytes or bone marrow cells.

A third object of the invention is the description of a high titerretroviral drug delivery system wherein one or more drugs are deliveredand may include tumor necrosis factor, either the prohormone or hormone,interleukin-2, or proteins that confer drug resistance including theprotein denoted, the multiple drug resistance protein (MDR).

A fourth object of the invention is the description of a retroviral drugdelivery system wherein a retrovirus carries DNA that encodes a highlycell secretable form of tumor necrosis factor.

A fifth object of the invention relates to a method of administering oneor more active protein(s) to a subject vertebrate host which comprisesadministering this drug delivery system either locally or systemically.Or, as alluded to above, the drug delivery system may be administered toa cell that is susceptible to viral infection, wherein the infectionoccurs in vitro, and the infected cell is then returned to the hostorganism where it produces the desired protein.

A sixth object of the invention relates to a method of delivering anactive protein to vertebrate host cells that require high titers ofretrovirus to effect infection of the cells thereby administering theprotein to the cells and consequently to the vertebrate host.

A seventh object of the invention relates to materials and processessignificant in the preparation of the above-described drug deliverysystem. These include proviral DNA comprising a DNA sequence encoding adesired active protein. The preferred molecules are those that can beused in cancer chemotherapy, such as tumor necrosis factor, IL-2,multiple drug resistant protein, etc. These sequences may be operablylinked to control sequences derived from a retrovirus, including apackaging site, and flanked by retroviral-derived LTRs, or to homologouscontrol sequences normally responsible for the expression of the proteindrugs.

In another embodiment, the general method of the invention can also becarried out by implanting Ψ-cells transfected with the proviral DNA ofthe previous paragraph, or cells infected with the pseudo virions theyproduce, to effect in situ production of the desired protein.Accordingly, the cotransfected Ψ-cells and cells infected with themodified viruses they produce provide pharmaceutical compositions whichare also aspects of the invention.

Also an aspect of the invention is a process to prepare the compositionsthereof which comprises isolating the delivery virions produced by theforegoing Ψ-packaging cells.

Brief Description of the Drawings

FIG. 1 shows the DNA sequence that encodes the gamma interferon signalpeptide, and a portion of the DNA sequence that encodes mature TNF.

FIG. 2 shows immunoprecipitation analysis of 35S-methionine labelled TNFencoded by pFVX TNF. Lane A, untransfected cells and lane B, cellstransfected with pFVX TNF. Methionine labels only the 26 kD precursor,not the 17 kD "mature" form (as predicted from the amino acid sequence).

FIG. 3 shows immunoprecipitation analysis of 35S-cysteine labelled TNFencoded by pFVX TNF. Lane A, untransfected cells and lane B, cellstransfected with pFVX TNF. Cysteine labels both the 26 kD precursor andthe 17 kD mature form (as predicted from the amino acid sequence).

FIG. 4 shows immunoprecipitation analysis of TNF retrovirus infected35S-cysteine labelled 3T3 cells. Lane A, supernatant derived from NIH3T3 cells; lane B, cytoplasmic lysates of NIH 3T3 cells; lane Csupernatant derived from cells infected with the FVX TNF retrovirus(TNF-V); and lane D, cytoplasmic lysates derived from cells infectedwith same. Note that the 26 kD form of TNF is found in the cytoplasmicextracts and is not secreted, whereas the 17 kD "mature" or secretedform is found in both the cytoplasmic extracts and the supernatants.

FIGS. 5A-5B show the structure and bioactivity of the TNF retroviralgenome. 5A: restriction map of FVX TNF retroviral genome; and 5B: plaqueassay of pFVXM transfected psi-am cells. The assay was performed asdescribed below.

Culture dishes with G418-resistant colonies were overlaid with L929cells at a density of 7.3×10⁴ cells/cm2. When the cells attached, after30 minutes, the medium was aspirated, and the cells were overlaid withDMEM supplemented with 10% FCS and 0.9% Noble agar. After incubation for18-24 hours, clones surrounded by a lysed zone of L929 cells wereisolated by cloning cylinders and expanded to mass culture.

FIG. 6 shows the construction strategy for infective drug deliveryretroviruses pLMDRL6, pLTNFL6 and pLIL-2L6.

FIG. 7 shows immunoprecipitation analysis of cells transfected orinfected with either the LTNFL6 plasmid or the high titer TNF retrovirusderived from LTNFL6. Lane A, PA317 cells transfected with the high titerretrovirus encoding both 26 kD and 17 kD TNF, LTNFL6; lane B, NIH 3T3cells uninfected; and lane C, NIH 3T3 cells infected with the high titerTNF retrovirus, LTNFL6 clone 8 present in the cell culture supernatantof LTNFL6 transfected PA317 cells.

FIG. 8 shows immunoprecipitation analysis of 35S-cysteine labelled humanmelanoma cells infected with the TNF retrovirus LTNFL6. Lane A, PA317cells transfected with LTNFL6; lanes B and C, uninfected and infected,respectively, NIH 3T3 cells with pLTNFL6 virus; and lanes D and E showuninfected and infected, respectively, human melanoma cells with thepLTNFL6 virus.

FIG. 9 shows PCR analysis of tumor infiltrating lymphocytes (TIL)infected with pLTNFL6 virus. Lane A, uninfected TIL; lanes B and C,infected human TIL; lane D PA317 transfected with pLTNFL6; and lanes E,F and G, 1:10, 1:100 and 1:1000 dilutions of same.

FIG. 10 shows the strategy for producing the two gene infective drugdelivery retrovirous that encodes the dominate selectable marker forneomycin resistance and TNF.

FIGS. 11A-11C: panel A, shows the restriction map of the cDNA sequencethat encodes 26 kD TNF. Panel B shows a hydrophobicity plot of 26 kDTNF, and panel C shows the DNA and amino acid sequences of the molecule.

FIG. 12 shows a restriction map of the cDNA sequence that encodes 26 kDTNF and the regions of the molecule that were deleted to produce thevarious muteins.

Table I shows the cytotoxic activity of various TNF constructs.

Modes of Carrying Out the Invention A. Definitions

As used herein, "drug delivery virion" refers to a modified retroviruswherein the genome is an RNA which contains control sequences derivedfrom retroviral nucleic acids operably linked to one or more codingsequences for an "active ingredient" protein(s). The genome is packagedin a protein envelope which is compatible with, and capable of causing"infection" with the contained genome in, a subject intended to betreated with the protein. The infection in this case extends only to theentry of the desired RNA into the cell and production of the protein; noadditional infective virions are produced.

High titer "drug delivery virions", or "high titer retrovirus" isintended to refer to those vectors known in the art that allowproduction of retrovirus at titers at least 10⁵ -10⁶ cfu/ml. Examples ofthis type of virus are shown in Bender, et al., above.

Thus, "dead-end" infection describes a modified form of infectionwherein the viral envelope facilitates the entry of the modified virusinto the cytoplasm, and the contained genome is expressed, but no newvirions are produced.

"Control sequences" refers to those nucleic acid sequences containing,for example, promoters and, often, enhancers which are necessary andsufficient for the production of the desired active protein ingredientby expression of the coding sequence operably linked to it.

In the case of the drug delivery retrovirions of the invention, the RNAgenome and its proviral counterpart also includes the Ψ packaging site.

"Tumor Necrosis Factor" or "TNF" as used herein refers to both nativeand recombinant forms of this known, mammalian cytokine. TNF has beenreferred to by other names in the literature, including "Cachectin" in"TNF-a". "Recombinant TNF" or "rTNF" refers to proteins, includingmuteins, produced by expression of recombinant DNA that have the same orsubstantially the same amino acid sequence as native TNF (or portionsthereof), and retain both the in vitro and in vivo biological activityof TNF. The isolation and production of both native and recombinantmammalian TNF, including human TNF, is known in the art. See, e.g.,Carswell et al., 1975, Proc. Nat'l Acad. Sci. USA, 72: 3666-3670;Williamson et al., 1983, Proc. Nat'l Acad. Sci. USA, 80: 5397-5401; Wanget al., 1985, Science, 228: 149-154; Beutler et al., 1985, J. Exp.Med.,161-984; Beutler et al., 1985, Science, 229:869; Beutler et al.,1985, Nature, 316:552; Pennicia et al., 1984, Nature, 312:724; Aggarwalet al., 1985, J. Biol. Chem., 260: 2345.

With regard to TNF, the terms "prohormone", and "mature" hormone" havethe following meanings. Prohormone refers to the 26 kd molecule, whereasmature hormone refers to the 17 kD molecule that results from theremoval of a 76 amino acid leader sequence. The prohormone is known tobe membrane bound and is not freely circulating, whereas the maturehormone is not membrane bound and is free to circulate. Thus,encompassed within the definition of TNF is the prohormone and maturehormones forms, and additionally, other forms of TNF wherein the 76amino acid leader sequence is removed and replaced with another leadersequence that facilitates the secretion of the mature hormone. A numberof leader sequences will perform this function, but preferred is thegamma interferon leader sequence, as described by Gray, P., et al.,1982, Nature, 295:503.

"Nucleic acid sequence" will sometimes be employed herein as a genericterm covering both DNA and RNA fragments. As the materials of theinvention include retroviral genomes and their proviral counterpart,particular functional sequences referred to will occur both in RNA andDNA form. The corresponding loci will be referred to interchangeably fortheir occurrences in both DNA and RNA, as it will be understood that inthe ordinary course of infection, such functionalities are, indeed,interchangeable. For example, the Ψ packaging site apparently isoperable in the RNA genome to be packaged; however, the correspondingsequences occur in the proviral DNA. Similarly, promoter, enchancer, andterminator sequence occur, though in slightly different forms, in boththe genomic RNA and proviral DNA forms. The interchangeability of thesefunctionalities in the various phase of the viral life cycle isunderstood by those in the art, and accordingly, rather looseterminology in regard to DNA or RNA status is often used in referring tothem. Specifically, sequences specified by a progression of bases shouldbe understood to include these specific sequences and their complements,both in DNA and RNA forms.

The pharmaceutical compositions of the invention include the drugdelivery virions produced by transfected intermediate cells which havebeen transformed with Ψ-helper provirus and thus produce emptyenvelopes. For simplicity in referring to these cells used in thepreparation of this composition, these cells will be referred to as"packaging" cells.

The resulting delivery virions can also be used to infect wild typecells in vitro, for example, as models for their ability to causeproduction of desired protein in the target host. These infected cellsare referred to herein as "tester" cells.

B. General Description

The crucial intermediate in the preparation of the compositions of theinvention is a proviral DNA vector containing the coding sequence forthe protein drug(s) to be administered. The preferred embodiment proteindrugs are those that are cytotoxic or cytostatic for tumor cells,preferably tumor necrosis factor (TNF) and interleukin-2 (IL-2), ordrugs that can be utilized in an effective chemotherapeutic regime, suchas multiple drug resistant protein. The DNA encoding such active proteiningredient may be obtained from any convenient source and, depending onthe protein chosen, can be synthesized chemically, recovered from a cDNAlibrary, isolated from genomic DNA, or otherwise obtained by means knownin the art.

The proteins to be administered according to the method of the inventioninclude any protein(s) which has a desired effect on an infected cell inthe subject to be treated. Advantages of the drug delivery system of theinvention are experienced especially when the protein operates withinthe cytoplasm of a target cell. For example, tumor necrosis factor (TNF)is capable of selectively killing tumor cells, but needs to transit thecell membrane to exert its effect. Other proteins, such as ribotoxinsand the various colony-simulating factors, also operateinteracellularly.

It will appreciated that retroviral vectors constructed to express twogenes may include two proteins that have prophylactic or therapeuticvalue, or one gene could express a dominate selectable marker whichwould facilitate identifying cells transfected with the two geneconstruct. An example of a selectable marker would be resistance to G418that is conferred on cells by the presence of the neomycin genesequences.

The system of the invention is applicable also to materials whosefunction is carried out outside the target cells or which function bybinding to receptors on the cell surface. In this case, however, thedrug delivery virions are administered indirectly as an implant oftransfected packaging cells or of tester cells which have been infectedwith the drug delivery virions. For example, tissue plasminogenactivator or urokinase, which act in the bloodstream itself, directly onsoluble enzymes in the blood, could be produced in situ by theseimplanted cells.

DNAs encoding the foregoing proteins are available in the art, and canbe obtained bracketed with linker sequences for convenient manipulation,if desired. The nature of the delivery system is such that both genomicand CDNA sequences can be used, since introns can be processed in theenvironment transfected by the provirus. The protein drug can be encodedin the delivery virion to specify any form of the protein desired, forexample, an active form, a mature form, a fused protein, a preprotein,or a preproprotein. In the example shown below, cDNA clones encodingTNF, IL-2, or MDR protein are used as the source of the coding sequence;however, clearly this is illustrative only, and any other desired codingsequence could also be employed. Further, it will be appreciated thattwo gene viruses can be constructed wherein one of the above moleculesis expressed along with a dominant selectable marker such as resistanceto G418.

The proviral transfer vector is obtained by isolation of an appropriateproviral form of a retrovirus such as the commonly used murine sarcomavirus (MSV), Moloney murine leukemia virus (MoMLV), Harvey sarcoma virus(HaSV), or a variety of other retroviruses. It is known that for certaintarget cells/tissues that they require a high tier of retrovirus tobecome infected. In these instances, the preferred retrovirus is onethat includes the gag sequences, the so called gag+vectors. Exemplary ofsuch vectors are those described by Bender, et al., above, and Miller etal., in Current Communications in Molecular Biology--Viral Vectors, page122, Cold Spring Harbor (eds. Gluzman, and Hughes, 1988). Since theproteins associated with the virion per se are deleted in theconstruction, even components derived from infectious retroviruses whichcause disease in humans, such as hepatitis, HTL VI, and LAVI, could alsobe used, although it is not necessary to utilize such materials which,of course, have the potential for psychological resistance among thesubjects to be treated. Further, infectivity host range could be alteredby introducing a foreign viral envelope glycoprotein gene (i.e., rabies,VSV, etc.) into either the recombinant provirus or the packaging Ψ typecell line.

The proviral form of the selected retrovirus is obtained by propagatingthe virus in tissue culture, isolating proviral DNA, cloning thisproviral DNA into a lambda phage cloning vector, and propagating therecombinant vector in a susceptible bacterial host where the phagevector is integrated. The proviral DNA is excised and reisolated. Theproviral DNA is then provided with suitable linkers and inserted into abacterial cloning vector for amplification. Suitable bacterial cloningvectors include pBR322, pML, or vectors the pUC series. These may needto be modified to eliminate or alter restriction sites and so forth, asis understood by those skilled in the art. The cloning vectors arerestricted and then provided with inserts of the linkerframed proviralDNAs.

The native proviral DNA inserts contain the viral protein encodingsequences flanked by long terminal repeat (LTR) sequences and containthe packaging site adjacent one of the LTRs. These protein-encodingsequences between the packaging site and the other LTR are theneliminated by appropriate restriction and/or exonuclease digestion andreplaced by linker or polylinker sequences. The appropriate sites may bealready available in the intervening region, or if not, these can beobtained using site-directed mutagenesis, as is understood in the art.

After amplification, the vectors containing the modified provirions arecleaved with suitable restriction enzymes to open the vectors forinsertion of the desired coding sequence. Since the control sequences,except for the packaging site, are in the long terminal repeats,insertion of a desired protein-encoding drug sequence into the linkerplaces it in operable linkage with the controls. The resulting modifiedvirion then becomes an expression system for the desired protein insteadof for the viral proteins, and still retains a packaging site to permitthis modified viral genome to be infective.

These drug delivery provirion DNAs are amplified and isolated usingknown techniques to provide a source of transfecting DNA for theΨ-packaging cells.

If desired, the inserted coding sequence in the modified virion, high orlow tier virion, can also include a marker sequence. If a two generetroviral vector is being used, one gene may encode neomycin resistanceand thus confer resistance to G418. If a significant decrease inexpression of the inserted sequence is observed then transfection of theΨ packaging cells can be coincident with transformation with vectorscontaining a suitable marker, most appropriately the G418 resistancemarker. Any suitable marker can, of course, be used, and such markerinclude, for example, Eco gpt conferring resistance to mycophenolic acidand DHFR sequence conferring methotrexate resistance.

The modified provirion, along with a marker plasmid, if necessary, istransfected into recipient packaging cells using standard transfectiontechniques such as calcium phosphate precipitation. The transformantsare grown in culture appropriate to their particular cell type; theΨ-3T3 cells illustrated below are cultured under conditions generallyused for wild type 3T3 cells. Of course, an appropriate amount of aselective component of the medium, such as G418, is also included toselect successful transformants.

The transformed packaging cells can be shown successfully to produce theproteins encoded by the inserted coding sequences in the modified virionby assessing the concentration of protein in the medium or cell lysate,as appropriate.

To obtain the packaged recombinant virions, supernatant from thepackaging cells is separated from the cells, for example, by using a0.45 micron filter. The virus is obtained from the filtrate by, forexample, high-speed centrifugation to harvest the viral particles or thefiltrate is used per se. The concentrated viral particles or thefiltrate are then formulated as pharmaceutical compositions.

In addition, the virion preparation can be assessed for competence toeffect drug delivery to target cells by using a tester cell line, forexample, the wild-type counterpart of the packaging cell line, whichproduces no empty viral capsules, or any cell line susceptible toinfection by the virus and, preferably, also, to the protein produced bythe recombinant virion. The amount of desired protein produced by thistester cell can be assessed, and, in the case of the appropriate cells,this assessment can be by the direct effect of the protein on the cells.

Both the packaging and tester cells can also be used as implants toprovide a source of the protein drug in situ.

It is important to note that the LTRs contain most of thetranscriptional control elements of retroviruses, including promotersand enhancers. Thus, while the inserted protein drug DNA sequences maybe transcribed under the control of viral control elements, it isintended that also encompassed within the invention are viral vectorsthat have the viral control elements replaced with promoters/enhancersthat normally regulate the transcription of a particular protein drug.

There are at least two basic approaches to obtain the expression ofvarious protein drugs wherein the DNA sequences that encode the drugsare under the control of their normal control elements.

The first approach entails the substitution of the promoter, or theenhancer and promoter, or just the enhancer in the 3' retroviral LTR ofthe provirus with a promoter, or an enhancer and promoter, or anenhancer from a heterologous virus or cellular gene. Upon transfection,such a provirus would express itself from the wild type retroviral LTR.After virus rescue due to the nature of the retroviral life cycle,mutations of the U3 region of the 3' LTR are immortalized in the 5' LTR.Thus, the substitution of a heterologous expression element in the 3'LTR becomes immortalized in the 5' LTR of the newly integrated provirus.Thus, the expression of the exogenous gene inserted into the recombinantretrovirus is driven from the heterologous expression element originallyresident in the 3' LTR of the recombinant provirus.

The second approach entails the deletion of the enhancer and promoter ormerely the promoter resident in the 3' LTR by either restrictionendonuclease digestion or through the application of site directedmutagenesis. When such a vector is rescued as an infectious retrovirusthe resultant infectious provirus lacks expression elements in both the5' and 3' LTRs. In this case, the expression defect is complemented bythe insertion of an exogenous promoter or promoter/enhancer combinationor a promoter enhancer combination plus an additional cis acting element(such as the U5 element of HTLV-1) between the LTRs of the recombinantprovirus, just 5' to the gene to be expressed. Under thesecircumstances, after infection and gene transfer, the transferred geneis now subject to the regulatory controls imposed by the proximalpromoter. Deletion of the expression elements in the LTRs insures thatthere is no interference from the LTR promoters.

Using the above approaches, retroviruses can be produced that have avariety of promoters, including the IL-2 promoter or the IL-2 receptorpromoter driving the transcription of their corresponding proteins.

C. Utility and Administration

The drug delivery system of the invention is effective in the net resultof transmitting protein drugs into cells where they may exert theireffects. The transfer occurs by virtue of viral infection so it ismerely necessary to juxtapose the drug delivery virions with the targetcells. If the target cells are localized in, for example, a solid tumorthe composition of the invention may be injected directly into the solidtumor. If the cells are, however, widely distributed such as in aleukemia or where, for example, red blood cells or bone marrow cells areneeded to be targeted, systemic intravenous injection is required.

Alternatively, the drug may be delivered by infecting the cells in vitroand returning the infected cells to the host organism where they expressthe protein drug. The latter procedure will be particularly useful forgene therapy involving particular types of cells, including bone marrowand skin fibroblast cells.

Notable applications of the drug delivery systems described herein willinvolve the infection of tumor infiltrating lymphocytes, or otherfunctionally similar cell types that act as vehicles for carrying theprotein drugs to tumor cells, with retroviruses that encode a variety ofprotein drugs that are effective as anti-cancer agents. Preferably theprotein drugs are IL-2 or TNF. Muteins of TNF that are membrane boundand cytotoxic are most preferred. For example, as will be describedbelow the TNF deletion muteins of prohormone TNF Δ(1→12) and TNF Δ(1+12)are transmembrane proteins and thus are firmly affixed to the cellsurface. Both are cytotoxic. Either of the muteins may be expressed intumor infiltrating lymphocytes (TIL). If the cells selected are TILs,they will be highly cytotoxic because of their endogenous tumor cellcytotoxic activity, plus the cytotoxicity attributable to the TNFmutein. When used in this format, it will be appreciated that an addedadvantage associated with the TNF mutein Δ(1→12) is that because it ismembrane bound and not released into the extra cellular environmentthere is little or no nonspecific cytotoxicity. A similar advantage isassociated with the Δ(1+12) mutein. Although a molecule of about 17 kDis released from the mutein, it is not cytotoxic.

The virions are prepared for injection in typical ways suitable foradministration of drugs by suspension in isotonic saline or othersuitable pharmaceutical excipient as is known in the art.

Implantation of packaging or tester cells is conducted by formulatingthem into suitable compatible formulations, such as physiologicalsaline, and directly injecting them into the desired location. The cellscan also be formulated using encapsulation techniques. (See, e.g., U.S.Pat. No. 4,391,909.)

D. Standard Methods

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described by Cohen, S. N., Proc. Natl. Acad. Sci.(USA) (1972) 69:2110, or the RbCl₂ method described in Maniatis et al.,Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Press,p. 254 was used for procaryotes or other cells which contain substantialcell wall barriers. For mammalian cells without such cell walls, thecalcium phosphate precipitation method of Graham and Van der Eb,Virology, 1978, 52:546 is preferred.

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniqueswhich are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

Site specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. See,e.g., New England Biolabs, Product Catalog. In general, about 1 μg ofplasmid or DNA sequence is cleaved by one unit of enzyme in about 20λ ofbuffer solution; in the examples herein, typically, an excess ofrestriction enzyme is used to insure complete digestion of the DNAsubstrate. Incubation times of about one hour to two hours at about 37°C. are workable, although variations can be tolerated. After eachincubation, protein is removed by extraction with phenol/chloroform, andmay be followed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol and resuspension in 10mM Tris, 1 mM EDTA, pH 7.5. If desired, size separation of the cleavedfragments may be performed by polyacrylamide gel or agarose gelelectrophoresis using standard techniques. A general description of sizeseparations is found in Methods in Enzymology , 1980, 65:499-560.

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 minutes at 20° to 25° C. in 50 mM Tris pH 7.6, 50 mMNaCl, 6 mM MgCl₂, 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fillsin at 5' sticky ends but chews back protruding 3' single strands, eventhough the four dNTPs are present. If desired, selective repair can beperformed by supplying only one of the, or selected, dNTPs within thelimitations dictated by the nature of the sticky ends. After treatmentwith Klenow, the mixture is extracted with phenol/chloroform and ethanolprecipitated followed by running over a Sephadex G-50 spin column.Treatment under appropriate conditions with S1 nuclease results inhydrolysis of any single-stranded portion.

Synthetic oligonucleotides are prepared by the triester method ofMatteucci et al., 1981, J. Am. Chem. Soc., 103:3185, or usingcommercially available automated oligonucleotide synthesizers. Kinasingof single strands prior to annealing or for labelling is achieved usingan excess, e.g., approximately 10 units of polynucleotide kinase to 0.1nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl2, 5 mMdithiothreitol, 1-2 mM ATP, 1.7 pmoles γ32P-ATP (2.9 mCi/mmole), 0.1 mMspermidine, 0.1 mM EDTA.

Ligations are performed in 15-30λ volumes under the following standardconditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP, 0.01-0.02(Weiss) units T4 DNA ligase at 4° C. (for "sticky end" ligation) or 1 mMATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for "blunt end"ligation). Intermolecular "sticky end" ligations are usually performedat 33-100 μg/ml total DNA concentrations (5-100 mM total endconcentration). Intermolecular blunt end ligations (usually employing a10-30 fold molar excess of linkers) are performed at 1 μM total endsconcentration.

In vector construction employing "vector fragments", the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) in orderto remove the 5' phosphate and prevent religation of the vector. BAPdigestions are conducted at pH 8 in approximately 150 mM Tris, in thepresence of Na⁺ and Mg⁺² using about 1 unit of BAP per μg of vector at60° C. for about 1 hour. In order to recover the nucleic acid fragments,the preparation is extracted with phenol/chloroform and ethanolprecipitated and desalted by application to a Sephadex G-50 spin column.Alternatively, religation can be prevented in vectors which have beendouble digested by additional restriction enzyme digestion of theunwanted fragments.

For portions of vectors derived from cDNA or genomic DNA which requiresequence modifications, site specific primer directed mutagenesis isused. This is conducted using a synthetic primer oligonucleotidecomplementary to a single stranded phage DNA to be mutagenized exceptfor limited mismatching, representing the desired mutation. Briefly, thesynthetic oligonucleotide is used as a primer to direct synthesis of astrand complementary to the phage, and the resulting double-stranded DNAis transformed into a phage-supporting host bacterium. Cultures of thetransformed bacteria are plated in top agar, permitting plaque formationfrom single cells which harbor the phage.

Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form; 50% will have the original sequence.The resulting plaques are hybridized with kinased synthetic primer at atemperature which permits hybridization of an exact match, but at whichthe mismatches with the original strand are sufficient to preventhybridization. Plaques which hybridize with the probe are then picked,cultured, and the DNA recovered. Details of site specific mutationprocedures are described below in specific examples.

More specifically, mutagenesis can be carried out using any number ofprocedures known in the art. These techniques are described by Smith,1985, Annual Review of Genetics, 19:423, and modifications of some ofthe techniques are described in Methods in Enzymology, 154, part E,(eds.) Wu and Grossman (1987), chapters 17, 18, 19, and 20. Thepreferred procedure is a modification of the Gapped Duplex site-directedmutagenesis method. The general procedure is described by Kramer, etal., in chapter 17 of the Methods in Enzymology, above.

Conventional M13 mutagenesis methods involve annealing a short syntheticoligonucleotide to single stranded M13 DNA having a cloned target codingsequence that is sought to be mutagenized. The oligonucleotide isalmost, but not entirely complementary to the target sequence and has atleast one mispaired nucleotide. After the annealing reaction, theremaining portion of the single stranded DNA must be filled in to giveheteroduplex DNA that can be transfected into a suitable host cell whichallows for the expression of the mutation. In the gapped duplex method,a partial DNA duplex is constructed that has only the target regionexposed, unlike the conventional methods which have the target regionand the rest of the single stranded M13 DNA exposed. Like theconventional methods, a short oligonucleotide is annealed to the targetregion, and extended and ligated to produce a heteroduplex. However,because only a small portion of single-stranded DNA is available forhybridization in the gapped duplex method, the oligonucleotide does notanneal to undesired sites within the M13 genome. Further, this methodhas the additional advantage of introducing fewer errors during theformation of the heteroduplex since only a very small region of DNA oneither side of the target region has to be filled in.

More specifically, the gapped duplex method involves cloning the targetDNA sequence into an appropriate M13 phage that carries selectablemarkers, such as for example the stop codon amber mutation. The latterallows for negative selection in a host cell that cannot suppress theeffects of the mutation. Preferably the phage is M13mp9 which containstwo amber codons in critical phage genes. Thus, the sequence thatencodes 26 kD TNF is cloned into M13mp9 amber+, and single stranded DNAis prepared therefrom using standard techniques. Next, double strandedreplicative form DNA from M13 GAP, a genetically engineered M13derivative that lacks the amber codons is cleaved with Hinc IIrestriction enzyme. The base sequence of M13 GAP is similar to M13mp18,which lacks both the amber codons and the sequence between base pairs6172 and 6323. This deletion flanks the multiple cloning sites of theM13mp series and generates a unique Hinc II site. Gapped duplex DNA isformed, using standard DNA/DNA hybridization techniques, consisting ofsingle stranded DNA having the amber codons, and a second strand of DNAfrom Hinc II digested M13 GAP lacking both the amber codons and the TNFcoding sequences. Thus, the only portion of the gapped duplex that isexposed is the 26 kD TNF target sequence. The desired oligonucleotide isannealed to the gapped duplex DNA, and any remaining gaps filled in withDNA polymerase and the nicks sealed with DNA ligase to produce aheteroduplex. The latter is transfected, preferably into a mismatchrepair deficient host, and mixed phage produced. From the mixed phagepopulation, phage carrying unmutated 26 kD TNF DNA, which also have theamber mutations, can be selected against by infecting the mixed phagepopulation into a host cell that cannot suppress the amber mutation.Clones can then be screened for phage that carry the desired TNFmutation.

Correct ligations for plasmid construction are confirmed by firsttransforming E. coli strain MM294 obtained from E. coli Genetic StockCenter, CGSC #6135, or other suitable host with the ligation mixture.Successful transformants are selected by ampicillin, tetracycline orother antibiotic resistance or using other markers depending on the modeof plasmid construction, as is understood in the art. Plasmids from thetransformants are then prepared according to the method of Clewell, D.B., et al., 1969, Proc. Natl. Acad. Sci. (USA), 62:1159, optionallyfollowing chloramphenicol amplification (Clewell, D. B., 1972, J.Bacteriol., 110:667). The isolated DNA is analyzed by restriction and/orsequenced by the dideoxy method of Sanger, F., et al., 1977, Proc. Natl.Acad. Sci. (USA), 74:5463 as further described by Messing et al., 1981,Nucleic Acids Res., 9:309, or by the method of Maxam et al., 1980,Methods in Enzymology, 65:499.

Host strains used in cloning and expression herein are as follows:

For cloning and sequencing, E. coli strain HB101 was used as the host.

For M13 phage recombinants, E. coli strains susceptible to phageinfection, such as E. coli K12 strain DG98 are employed. The DG98 strainhas been deposited with ATCC 13 July 1984 and has accession number 1965.

E. EXAMPLES

The following examples are supplied herewith as illustrative of theinvention and are not to be construed to limit the invention.

EXAMPLE 1 Preparation of Modified Provirion Containing TNF

The coding sequence for human TNF was obtained from the clone pB11, acDNA clone extensively described in U.S. Pat. No. 4,677,063, issued Jun.30, 1987, and incorporated herein by reference. It may also be derivedfrom clone pB11 also shown in U.S. Pat. No. 4,677,063. This clone isalso on deposit at ATCC, ATCC# 39894, deposited Oct. 15, 1984.

The plasmid pAW711, also there described, and deposited Nov. 8, 1984,ATCC# 39918 can also be used as a source of TNF sequences.

The vector containing the retroviral control system in proviral formalong with the packaging site is a derivative of pEVX; pEVX contains theLTRs and Ψ site from MoMLV inserted into the EcoRI site of pML(Kriegler, M., et al., 1984, Cell, 38: 483-491). pEVX was modified toeliminate a splice donor site by replacing a SmaI/BalI segment with a978 bp SamI/SmaI fragment from the Harvey sarcoma virus (HaSV), whichcontains the 3' portion of the 5' LTR and the 5' portion of the HaSVgenome. The resulting construct was digested completely with SstII andpartially with BglII to obtain a 669 bp fragment lacking nonessentialHaSV regions. This fragment was gel purified and religated to SmaI/BalIdigested pEVX. The resulting vector pFVXM contains a polylinker betweenthe LTR fragments derived from MoMLV and includes the packaging sitefrom this virus, but lacks the splice donor site in the upstream LTR.pFVXM, on deposit with the American Type Culture Collection, AccessionNo. 67,103.

The pFVXM vector is amplified in E. coli strain Hb101, and the plasmidDNA isolated. pB11 is likewise amplified in E. coli HB101 and reisolatedas plasmid DNA. Both preparations of plasmid DNA are treated with Pst I(to excise the TNF-encoding region from pB11 and to open pFXVM in thepolylinker region). Ligation of the fragments is carried out usingstandard conditions and the ligation mixture transformed into E. coliHB101 to Amp_(z) ^(R). Plasmid DNA was again isolated, and the correctorientation of the insert was established by restriction analysis.Recombinant plasmids with the correct orientation of the TNF-encodingsequences are designated pFVXMTNF, and used to transfect appropriatepackaging cells as described below. One such vector, pFVXMTNF6, isreferred to more in detail below.

In a similar manner, for example, pFVXM may be digested with Pst I andligated to DNA sequences encoding ricin A toxin, CSF-1, and urokinase,each provided with suitable Pst I linkers when needed. Furtherdesirable, are vectors that encode leukocyte interferons, preferablyhybrid interferon molecules as described by Weck et al., in NucleicAcids Res., 1981, 9(22): 6153, and in U.S. Pat. No. 4,678,751, toGoeddel D.V.N. Particularly preferred is the hybrid interferondesignated LeIF-AD (Bgt). The resulting vectors are designated pFVXMRA,pFVXMCSF, pFVXMUK and pFVXMIF, respectively. The DNA sequence thatencodes ricin A toxin is described in U.S. patent application, Ser. No.06/837,583, while the cDNA sequences that encode mCSF, and urokinase aredescribed in U.S. Pat. Nos. 4,847,201, 4,868,119, and EPO 92,182 and inJacobs et al., 1986, DNA, 4(2): 139-146, respectively.

It is will also be understood that the TNF sequence present in pFVXMTNFmay have the 76 amino acid leader sequence associated with theprohormone partially deleted, or replaced with another leader sequencethat produces abundant amounts of the nature form of TNF. For example,this construct can be produced by removing the TNF Pst I fragment frompFVXMTNF and subcloning it into a suitable M13 vector, followed bymutagenesis with an appropriate oligonucleotide.

Thus, the DNA fragment encoding 26 kD TNF was excised from pFVXMTNF, andmutagenized after sub-cloning into M13mp19 amber using the followingoligonucleotide (Cetus number CP 383) that encodes the gamma interferonsignal peptide:

5'-TCGAGAAGATGATCTGACGCCAAGAGAACCCAAAACGATGCAGAGCTGAAAAGCCAAGATATAACTTGTATATTTCATGGTGTCCTTTCCAGGGG-3'

The mutagenized construct containing the γ-interferon signal peptide wasexcised from the M13 vector, and cloned into a derivative of pFVXMtermed pUC.FVXM Δ HIII to produce pUC.FVXM ΔHIII TNF γ sig. The latterconstruct is on deposit with the American Type Culture Collection withAccession Number 68121, and has been further deposited with Cetus MasterCulture Collection, and is denoted 3688. The DNA that encodes the matureform of TNF with the γ-interferon signal peptide is shown in FIG. 1;only a portion of the DNA that encodes the 17 kD molecule is depicted.Shown below is the γ-interferon signal peptide linked to 17 kD TNF.##STR1##

pUC.FVXM ΔHIII is similar to pFVXM with the exception that it lacks 1110base pairs upstream of the 5'-LTR. Immunoprecipitation followed bySDS-PAGE of cell extracts, both lysates and supernatants obtained fromNIH-3T3 cells transfected with pUC.FVXM Δ HIII TNF γ sig. revealed nodetectable 26 kD TNF, but considerable amounts of the 17 kD molecule.

In addition to 17 kD or 26 kD TNF, retrovirions can be constructed thatencode muteins of these molecules. The preferred muteins are those thatmaintain TNF in a membrane bound, cytotoxic form. More preferred aremuteins that have deleted the first 12 amino acids, TNF Δ(1→12), or thefirst and twelfth amino acids, TNF Δ(1+12), of the mature form of TNF(i.e. 17 kD TNF). In contrast to these two muteins, it was determinedthat TNF Δ(1+13) is membrane bound but not cytotoxic. It was thusemployed as a control in some of the examples presented below. Thenumbering of the amino acids corresponds to the amino acid sequence ofthe prohormone form of TNF shown in FIG. 11. The muteins can begenerated using standard mutagenesis techniques and the followingoligonucleotides to perform the mutagenesis.

    ______________________________________                                        CP 467: Δ (1 → 12)                                               5'-TAC AAC ATG GGC TAC TGC CTG GGC CAG AGG-3'                                 CP 472:                                                                              screens Δ (1 → 12)                                               5'-TGG GCT ACT GCC TGG G-3'                                            CP 495: ΔVAL 1                                                          5'-TCG AGA AGA TGA TCT TGC CTG GGC CAG AGG-3'                                 CP 496:                                                                              screens ΔVAL 1                                                          5'-TGA TCT TGC CTG-3'                                                  CP 497: ΔPRO 12                                                         5'-TAC AAC ATG GGC TAC CTT GTC ACT CGG GGT-3'                                 CP498: screens ΔPRO 12                                                         5'-GGC TAC CTT GTC-3'                                                  CP 499: ΔVAL 13                                                         5'-TGC TAC AAC ATG GGC AGG CTT GTC ACT CGG-3'                                 CP 500:                                                                              screens Δ13                                                             5'-ATG GGC AGG CTT-3'                                                  ______________________________________                                    

Briefly, the oligonucleotides are kinased using the following reactionsolution and conditions: 3 μl 10×KB buffer, 3λ10 mM rATP (1:10 dilutionof 0.1M rATP stock), 2λ mutagenic oligonucleotide (100 pmole/λ), 21λ H₂O, and 1λ polynucleotide kinase (10 units/λ). The reaction is run at 37°C. for 45 minutes, and then at 65°-68° C. for 5 minutes. Next, 24λ ofthe kinased oligonucleotide is diluted with 56λ of H₂ O to give 2pmole/λ.

The gapped duplex is formed as described below, followed by annealingthe oligonucleotides. The following reagents are combined in a totalvolume of 40λ:8λ5×GDB buffer, 0.50 pmole ssDNA, and 0.10 pmole Hinc IIlinearized M13 GAP RF DNA. 10λ is removed for future use, and theremaining 30λ is treated sequentially as follows: 100° C. for 3 minutes,65° C. for 5 minutes, followed by cooling to room temperature for 30minutes, and then placing the reaction mixture on ice. Next, 10λ ofgapped duplex and 10λ of control ungapped material is subject toelectrophoresis on a agarose gel to check gapped duplex formation.Assuming the gel shows the presence of a third band, the gapped duplexhas formed and the kinased oligonucleotides can be annealed to theduplex by combining 16λ of gapped duplex reaction mixture, and 4λ ofdiluted kinased oligonucleotide, and heating the mixture to 65° C. for 3minutes, followed by cooling to room temperature for 20 minutes.

To produce TNF Δ(1+12) and TNF Δ(1+13), two kinased oligonucleotideswere annealed to the same gapped duplex. CP 495, CP 497, and CP 499 werekinased as before, but diluted to a concentration of 4 pmole/λ. Toproduce TNF Δ(1+12), 2λ of CP 495 and 2λ of CP 497 was added to 16λ ofgapped duplex and annealed. To produce TNF Δ(1+13), 2λ of CP 495 and 2λof CP 499 was added to 16λ of gapped duplex and annealed.

The heteroduplex is completed by the appropriate extension and ligationreactions consisting of combining the following reagents in a totalvolume of 40λ:10λ gapped duplex and primer, 4λ10×PEL buffer, 4λ dNTP's(0.25 mM solution made from 10 mM stocks, 3λ ATP (10λ of 0.1M ATPstock+1490λ H₂ O=0.662 mM), 17λ H₂ O, 1λ Klenow (5 u/λ), and 1λ T4 DNAligase (0.6 Weiss u/λ, diluted stock with 1×PEL). The reaction isconducted at 16° C. for 2 hours, followed by transformation of 10λ ofthe extension/ligation mixture into 200λ of thawed competent HB2154cells. The cells are kept at 0° C. for 30 minutes, and then 42° C. for1.5 minutes, followed by plating various volumes of the transformationmix (e.g., 50λ , 10λ, etc.) with 100λ of fresh overnight culture ofHB2151 cells+3.0λ of soft agar.

The resulting plaques are screened using the plaque hybridizationprocedure. While a variety of such procedures are known, a descriptionof the preferred procedure follows. Plates are replicated onto duplicatenitrocellulose filter papers (S & S type BA-85) and the DNA fixed to thefilter by sequential treatment for 5 minutes with 0.5N NaOH plus 1.5MNaCl; 1.0M NaCl plus 0.5M Tris-HCl pH 7.4; and 2×SSC (standard salinecitrate). Filters are air dried and baked at 80° C. for 2 hours, invacuo.

The duplicate filters are prehybridized at 55° C. for 2 hours with 10 mlper filter of DNA hybridization buffer, 5×SSC, pH 7.0, 5×Denhardt'ssolution (polyvinylpyrrolidone, plus Ficoll and bovine serum albumin;1×0.02% of each), 50 mM sodium phosphate buffer at pH 7.0, 5 mM EDTA,0.1% SDS, and 100 μg/ml salmon sperm DNA. The prehybridization buffer isremoved and the samples hybridized with the appropriate kinased probe,that is to say kinased oligonucleotides as shown above, under conditionswhich depend on the stringency desired. About 2×10⁶ cpm/ml total isused. Typical moderately stringent conditions employ a temperature of42° C. plus 50% formamide for 24-36 hours with 1-5 ml/filter of DNAhybridization buffer containing probe. For higher stringencies hightemperatures and shorter times are employed. The preferred hybridizationconditions consists of hybridizing the probes to the filters in 5×SSC,Denhardt's solution, 50 mM NaPO4, pH 7.0, 5 mM EDTA, 0.1% SDS, and 100μg/ml salmon sperm DNA at 100 below the TM of the oligonucleotide usedto do the screening. Next, the filters are washed twice, 30 minutes eachwash, at room temperature with 2×SSC, 0.1% SDS, then washed once with2×SSC and 0.1% SDS at 5° C. below the TM of the oligonucleotide used toscreen, and air dried. Finally, the filters are autoradiographed at -70°C. for 36 hours. Autoradiography reveals those plaques containing thevirus that carries the muteins of interest.

To screen for double mutants, one set of filters were probed with onescreening oligonucleotide and a replicate set of filters (lifted fromthe same plates) were probed with the other appropriate screeningoligonucleotide. The resultant autoradiographs were aligned and plaquesthat hybridized to both screening oligonucleotides were picked andsequenced.

The mutagenized constructs containing the various TNF muteins areexcised from the M13 vectors, and cloned into one of several possiblevectors to produce infectious retrovirions. Exemplary vectors includepFVXM, pUCFVXM ΔHIII, high titer retroviral vectors, preferably pLNL6described in Example 5, or vectors that yield two gene retrovirions.preferably pLNSX, pLXSN and pLNCX as described in Example 7. The latterthree vectors all carry the neomycin gene sequences.

EXAMPLE 2 Production of Drug Delivery Retrovirions that encode TNF

The pFVXM-TNF prepared as described in Example 1 (10 μg) is mixed with 1μg of pSV2-NEO (Southern et al., 1982, J. Mol. Appl. Gen., 1: 327-341),which contains the marker sequences conferring resistance to theantibiotic G418. Transfection was conducted using a modification of thecalcium phosphate method of Wigler, et al., 1978, Cell, 14: 725.Briefly, 10 μg of carrier DNA, diluted with sterile 1 mM Tris, pH 8.1,0.1 mM EDTA, was added to 100 mm Petri dishes, along with plasmid DNA,50-1,000 ng per 100 mm Petri dish, followed by the addition of 2.5MCaCl₂. This mixture was agitated thoroughly to assure uniformsuspension, and an equal volume of 2X HEPES (N-2-hydroxyethyl diperazineN'-2-ethanesulfonic acid) buffered saline, pH 7.1, was added. Thismixture was also agitated to assure uniform suspension, after which aprecipitate was allowed to form. Thirty minutes later, 1 ml of thesuspension was added to psi AM cells in 100 mm Petri dishes containing10 ml of DMEM supplemented with 10% fetal calf serum. The cultures wereincubated at 37° C. for 16 hours and subsequently the medium replacedwith fresh growth medium. Next, the growth medium was replaced againwith fresh medium, but supplemented with 400 μg/ml of G418, obtainedfrom Gibco. After an initial growth period, the cells were grown onselection medium containing 400 μg/ml G418 and resistant colonies werepicked, and transferred to 24-well tissue culture dishes for testing forTNF production.

The cellular proteins were labelled with either ³⁵ S-cysteine or ³⁵S-methionine. The cells were first cultured on DMEM lacking cysteine ormethionine, but containing 5% dialyzed fetal calf serum, for 30 minutesat 37° C. to effect cysteine or methionine starvation. One hundred μCiof ³⁵ S-cysteine or ³⁵ S-methionine having a specific activity ofapproximately 400 Ci/mmol was added and the cells further incubated for2 hours at 37° C. The supernatant was removed and saved. The cells werelysed with lysis buffer and the lysate supernatant was also recovered bycentrifugation. Both the clarified lysate and culture supernatant weretested for the presence of TNF as follows.

Polyclonal antisera to recombinant TNF prepared in rabbits were added toeach test material in a centrifuge tube and incubated at 4° C. for 1hour with shaking. This was followed by the addition of a 50% suspension(v/v) of protein A attached to Sepharose CL4B and followed by incubationat 4° C. for 30 minutes.

The beads were pelleted in a microfuge and washed. The precipitatedmaterial was removed from the beads by boiling in SDS. The solubilizedTNF-containing solutions were loaded onto 12.5% polyacrylamide gel forelectrophoresis and the proteins were fixed and stained as well as readby autoradiography. The results are shown in FIGS. 2 and 3.

FIG. 2 shows the results of labelling with ³⁵ S-methionine. Labelappears only in the leader sequence of the 26 kD unprocessed protein inthe lysate; no label is present in the mature, 17 kD, secreted form(which does not contain methionine residues).

FIG. 3 shows the results of ³⁵ S-cysteine labelling; both theunprocessed and mature forms are labelled, as expected.

Cell supernatants were also assayed for TNF using the L-929 assay below.The ability of these supernatants to show TNF activity was completelydestroyed by preincubation with the rabbit anti-TNF antisera.

EXAMPLE 3 Recovery of Drug Delivery Virions

The supernatants from the cells of Example 2 which secrete TNF into themedium were filtered through 0.45 micron Millipore filters to ensurethat no cells were transferred. Similarly, one can centrifuge thesupernatants at 3,000×g to pellet any cells or cellular debris. Thesupernatant contains the recombinant virion designated TNF-V.

EXAMPLE 4 Dead-End Infection of Tester Cells

The TNF-V prepared in Example 2 was used to infect 1×10⁵ NIH 3T3 or RAT2cells by incubation with 4 μg/ml of polybrene at 37° C. overnight in aCO₂ incubator. Cell supernatants and lysates were analyzed for TNFproduction using ³⁵ S-cysteine labelling, immunoprecipitation andradioautography exactly as described above in Example 2. The results forinfected cells are shown in FIG. 4. Both the 17 kD and 26 kD forms ofTNF contain label.

Supernatant from these cells also showed TNF activity using the L-929cytotoxicity assay, which activity was removed by incubation with rabbitanti-TNF antisera.

Assay for TNF Activity

FIG. 5 shows the structure and bioactivity of the TNF retroviral genome.Lane A, restriction map of FVX TNF retroviral genome, and lane B, plaqueassay of pFVXM transfected psi-am cells. To assay biological activity ofthe TNF, the L-929 assay system was used. The L-929 cells are preparedovernight as monolayers in microtiter plates. The test samples arediluted 2-fold across the plate, UV irradiated, and then added onto theprepared cell monolayers. The culture media in the wells are thenbrought to 1 μg/ml actinomycin D. The plates are allowed to incubate 18hr at 37° C. and the plates are scored visually under the microscope.Each well is given a 25, 50, 75 or 100% mark signifying the extent ofcell death in the well. One unit of TNF activity is defined as thereciprocal of the dilution at which 50% killing occurs.

In addition, a more sensitive version of this assay was developed thatmonitors the release of ³⁵ S labelled peptides from prelabelled cells,when treated with the test sample and actinomycin D. This version of theassay can be used to quantitate potency, e.g., to evaluate the relativepotency of oocyte translated material. Briefly, actively growing L-929cultures are labelled with ³⁵ S methionine (200 μCi/ml) for 3 hours inmethionine-free media supplemented with 2% dialyzed fetal calf serum.The cells are then washed and plated into 96 well plates, incubatedovernight, and treated the next day with 2-fold dilutions of testsamples and 1 μg/ml actinomycin D. The cultures were then incubated at37° C. for 18 hours. One hundred λ supernatant aliquots from each wellwere then transferred onto another 96 well plate, acid (TCA)precipitated, and harvested onto glass fiber filters. The filters werewashed with 95% ethanol, dried and counted. An NP₄₀ detergent control isincluded in every assay to measure maximum release of radioactivity fromthe cells. The percent ³⁵ S release is then calculated by the ratio ofthe difference in count between the treated cells and untreated controlsdivided by the difference between NP₄₀ treated cells and untreatedcontrols, i.e., by the ratio: ##EQU1## Higher TNF potency results inhigher values of this ratio.

EXAMPLE 5 Preparation of Modified High Titer Provirion ContainingProtein Drug Encoding Sequences

The coding sequences for human TNF, IL-2, and MDR were cloned into thehigh titer retroviral vector, pLNL6. The vector is described by Bender,et al., 1987, J. of Virol., 61(5): 1639-1646. FIG. 6 shows theconstruction strategy for infective drug delivery retroviruses pLMDRL6,pLTNFL6 and pLIL-2L6.

Isolation of MDR Sequence/pLMDRL6

The initial step in the production of pLMDRL6 was the isolation of theDNA sequence that encodes MDR. The DNA sequence that encodes MDR isdescribed in U.S. patent application, Ser. Nos. 892,575, and 845,610,and also in PCT/US87/00758. The full length cDNA sequence that encodesthe MDR protein is shown in Table 5 of the PCT application. It wasinitially cloned into a commercially available vector, pGEM3Z(f-), as aXba I-Cla I fragment. To accomplish this, Cla I-Hind III linker adaptorswere ligated to the Cla I-Xba I MDR fragment. This construct was thenligated into the Xba I-Hind III site of pGEM3Z(f-) resulting inpGEM3Z(f-)MDR.

Using pGEM3Z(f-)MDR, the high titer vector pLMDRL6 was produced asfollows. Fifty μg of pGEM3Z(f-)MDR was digested with 200 units of Cla I(New England Bio Labs) in Tris-acetate buffer consisting of 33 mMTris-acetate, pH 7.9 (66 mM potassium acetate, 10 mM magnesium acetate,0.5 mM dithiothreitol) in a total volume of 500λ. The reaction wasconducted at 37° C. for 60 minutes after which the DNA was phenolextracted and ethanol precipitated in an ethanol solution containing2.5M ammonia acetate. The precipitate was resuspended in 400λ of waterand the Cla I digested pGEM3Zf(-)MDR subjected to a second digestionwith Eco RI as follows.

To the 450λ of ethanol precipitated and resuspended material was added50λ of 10×Tris acetate buffer and 38 units of Eco RI (New England BioLabs). The mixture was incubated at 37° C. for 60 minutes, and thematerial phenol extracted, and ethanol precipitated as described above.The ethanol precipitate was resuspended in 100λ of Tris-EDTA buffer (10mM Tris (pH 8.0) 1 mM EDTA). A sample was prepared for electrophoresisby adding 20λ of loading buffer consisting of 0.25% bromophenol blue,0.25% xylene cyanol (15% Ficol) type 400 in water. The sample waselectrophoresed in a 1% agarose preparative gel in Tris acetate bufferconsisting of 40 mM Tris-acetate, pH 8.0 (2 mM EDTA) electrophoresisbuffer.

Following electrophoresis, a gel fragment containing the 4.2 kb MDR EcoRI-Cla I fragment was excised from the gel, and the DNA isolated usingstandard glass bead purification techniques.

Preparation of pLMDRL6

The high titer retrovirus vector, pLNL6 was prepared to receive the 4.2kb MDR Eco RI-Cla I fragment as follows. The procedures employed weresimilar to those used to insert the fragment into the pGEM3Z(f-) vector.Briefly, 50 μg of pLNL6 was digested with Cla I and subsequently with 38units of Eco RI. This produced a 4.7 kb Eco RI-Cla I fragment which wasisolated using standard electrophoresis techniques. Next, the 4.2 kb MDREco RI-Cla I fragment and the 4.7 kb Eco RI-Cla I pLNL6 fragment wereligated to produce the high titer MDR expression vector, pLMDRL6. Theprocedure consisted of ligating 1 μg of the MDR Eco RI Cla I fragment to1 μg of the 4.7 kb Eco RI-Cla I pLNL6 fragment in a total volume of 25λin Tris-acetate buffer containing 4 mM ATP and 400 units T4 DNA ligase(New England Bio Labs). The reaction was run for 16 hours at 4° C.,after which the ligation mixture was diluted 1:4 with TE buffer. Five λof this mixture was transformed into competent DH5α cells (BethesdaResearch Labs). Eight clones were picked, grown up overnight, andanalyzed for the presence of MDR inserts as follows.

One ml of an overnight of each of the eight clones was centrifuged in amicrofuge, supernatants removed, and the bacterial pellets cracked usinga cracking buffer consisting of 200λ phenol, 170λ TE buffer, and 30λ6×loading buffer. The samples were vortexed, centrifuged for 5 minutes,and the supernatants electrophoresed on a 0.8% agarose gel in TAE bufferat 50 volts for 16 hours. The slowest migrating clones were chosen forfurther analysis, and one of the clones, clone 5 was selected forfurther analysis by miniprep restriction digestion, and based on theresults of the restriction analysis, was shown to carry the plasmidpLNL6.MDR.

Production of Amphotropic MDR1 Virus

Using the pLMDRL6 vector, amphotropic virions containing the MDRsequence were produced as follows. About 4×10⁵ PSI-2 cells were platedin 100 mm tissue culture dishes and 16 hours subsequently, 3 hoursbefore transfection of the vector pLMDRL6 into PSI-2 cells, the PSI-2cells were refed fresh media, and the cells transfected via calciumphosphate mediated gene transfer.

PA317 cells were infected with media containing virions harvested fromPSI-2 cells as follows. 5×10⁵ PA317 cells were seeded per 60 mm tissueculture plate, and the cells allowed to grow overnight. The media fromtransfected PSI-2 cells was removed, and the cells refed 4 ml of freshmedia. The PSI-2 cells were allowed to secrete virions into the mediaovernight, after which the media was harvested, and spun at 8,000 rpmfor 5 minutes. The supernatant was used to infect the PA317 cells.Infection consisted of removing the cell culture media from the PA317cells, and incubating them with 2 ml of the PSI-2 supernatant in thepresence of 4 μg/ml polybrene. Twenty four hours later, the PA317 cellswere subcultured and seeded at 3-4×10⁴ cells/100 mm tissue cultureplate. These cells were then selected for multiple drug resistance 48hours after subculture by exposure to 20 ng/ml of vinblastine. 7 to 10days later, colonies were apparent. None were apparent in theuntransfected controls. Individual colonies were picked and found to beresistant to 20 ng/ml of vinblastine in continuous passage. Supernatantsderived from such colonies were found, upon infection of normally drugsensitive cells, to confer drug resistance with a titer of 1×10⁵ cfu/ml.

pLTNFL6

The TNF coding sequence used to produce pLTNFL6 was obtained fromplasmid B11 described in U.S. Pat. No. 4,677,063. The TNF sequence ispresent on a Pst I fragment, and thus was removed by Pst I digestion andinserted into the Pst I site of the commercially available vector pGEM-3(Promega), to produce pGEMTNF14.

Preparation of pLTNFL6

The vector pLTNFL6 was produced in several steps. Firstly, the MDRsequence in the vector pLMDRL6, described above, was excised, as was thesequence from the vector pGEMTNF14 that encodes TNF. The vector fragmentlacking the MDR sequence and the TNF sequence were blunt ended andligated to produce pLTNFL6.

In more detail, what this entailed was excising the MDR sequence bydigesting 50 μg of pLNL.MDR in TA buffer with 200 units BamHI, (NewEngland Bio Labs), 200 units Hind III (New England Bio Labs), and 90units Cla-I in a final volume of 500λ for 2 hours at 37° C. Followingthis incubation period, the vector fragment lacking the MDR codingsequence was blunt ended by adding 10λ of 10 mM dNTP (dATP, dCTP, dGTP,and dTTP), and 63 units of T4 DNA polymerase (Promega) and the mixtureincubated for an additional 20 minutes at 37° C., followed by furtherincubation for 10 minutes at 68° C. Next, the reaction digest was phenolextracted, ethanol precipitated, and electrophoresed on a 1% agarosegel, using essentially the procedures described previously. The 4.7 kbblunt ended fragment was excised from the gel and isolated usingstandard glass bead purification procedures.

Similarly, 50 μg of pGEM TNF14 was digested in TA buffer with 200 unitsof Pst I in a final volume of 500λ for 2 hours at 37° C. The reactiondigest was treated as described above, and fractionated on a 1% agarosegel. A 1.1 kb encoding fragment that carries the TNF encoding sequencewas excised from the gel and isolated as described above using glassbeads. This fragment was blunt ended as follows. One μg of the fragmentwas incubated in 30λ of TA buffer containing 0.05 mM dNTP's and 0.15units T4 DNA polymerase. The mixture was incubated for 20 minutes at 37°C., and subsequently for an additional 10 minutes at 68° C. The bluntended fragment was purified using glass beads, and resuspended in waterin preparation for ligation to the blunt ended 4.7 kb vector fragment.

The ligation reaction was conducted as follows. 0.8 μg of the 1.1 kbblunt ended TNF fragment was combined with 1.0 μg of blunt ended 4.7 kbvector fragment in a total volume of 25λ of TA buffer. The buffercontained 4 mM ATP and 400 units T4 DNA ligase (NEB). Ligation wasconducted for 16 hours at 4° C., after which the ligation mixture wasdiluted 1:4 with TE buffer, and then 5λ of the mixture was transformedinto 100λ of competent DH5α cells. Next, various dilutions of thetransformed cell mixture were plated onto nitrocellulose filters, andincubated on culture plates containing 50 μg/ml of ampicillin. After 16hours at 37° C., the plates were replica plated using a second set ofnitrocellulose filters. The new nitrocellulose filters served as masterplates. The original filters were processed in order to fix thebacterial DNA to the filters using standard alkaline lysis procedures.Subsequently, colonies containing TNF were identified using an endlabelled oligonucleotide probe having the sequence:

5'-TCA GCT CCA GCC CAT TGG-3'

Hybridization consisted of preincubating the filters in 5×SSC,4×Denhardts, 50 mM NaPO4, pH 6.8, 0.1% sodium dodecyl sulfate, and 100μg/ml salmon sperm DNA. The filters were pre-incubated for 1 hour,followed by 1 hour incubation in 15 ml of the same solution containingkinased probe. Twenty picomoles of the probe was used, and the reactionwas conducted at a Tm of 55° C. with shaking at about 120 revolutionsper minute. Following the incubation period, the filters were washedwith decreasing concentrations of SSC solution as follows. The firstwash consisted of 6×SSC containing 0.1% SDS, the second wash 4×SSCsolution containing 0.1% SDS, and the third wash containing 2×SSCcontaining 0.1% SDS. The first and second wash was repeated twice withthe wash period being 15 minutes. The third wash was conducted one timealso for a 15 minute wash period. All of the washes were conducted at60° C. with 120 revolutions per minute agitation. Following washing, thefilters were air dried, and autoradiographed at -70° C. Based on theautoradiographic results, 8 clones were picked from the master platesand inoculated into 3.5 ml of R2 media supplemented with 200 μg/ml ofampicillin. The bacteria were grown up under standard conditions andplasmid DNA isolated and restricted with Eco RI to identify those thathave the TNF fragment inserted in the proper orientation. Eco RIdigestion was carried out in 31.5λ of 10×buffer (0.33M Tris-acetate, pH7.9; 0.66M potassium acetate; 0.10M magnesium acetate; 5 mMdithiothreitol), 2.5λ of RNase (10 mg/ml), 265λ of glass distilledwater, and 13λ of Eco RI (20 units/λ, New England Biolabs). Controlswere run wherein the reaction mixture lacked Eco RI. The reaction wasstarted by combining 1λ of the appropriate clone with 34λ of thereaction cocktail and subsequent incubation at 37° C. for 1 hour. Thereaction digests were run on 1% TEAE gels. Three of the clones, TNF3,TNF6, and TNF7 exhibited the expected fragments having the followingnumber of base pairs 3180, 1613, and 900. Thus, based on these results,which corresponds to the expected orientation for the TNF codingsequence, TNF3, which corresponds to the vector pLTNFL6, was selectedfor further study. TNF3 was expanded, and a large scale plasmidpreparation made and analyzed by restriction digestion using Eco RI toconfirm the orientation of the TNF coding sequences.

Transfection of PA317 cells with pLTNFL6

Transfection of PA317 cells was achieved by seeding 3×10⁵ cells per 100mm tissue culture dish containing 10 ml of media. The cells were allowedto grow for 24 hours, and then transfected by removing the media andadding the following solution: 10 μg of pLTNFL6, 1 μg of aβ-actin-neomycin vector 10 μg of sheared LTK-carrier DNA, and 500λ of2×HBS (10 mM KCl, 11 mM D-glucose, 1.4 mM Na2HPO4, 42 mMHepes, and 171mM, NaCl, pH 7.05). Water was added to make a final volume of 950λ.Next, 50λ of 2.5M CaCl2 was added, and the two immediately vortexed for15 seconds. After a precipitate had formed the tube was incubated for 30minutes at room temperature. The entire solution was then added to a 100mm tissue culture dish containing PA317 cells, and the dish incubatedovernight at 37° C. Subsequently, the media was changed with freshmedia, incubated for another 24 hours at 37° C. and the cellstrypsinized and plated at serial 1:4 dilutions into medium supplementedwith 400 μg/ml of G418. The media was changed every 3 to 4 days, and 15days after the transfection, clones were isolated using cloningcylinders, and the resulting clones grown up in mass culture and assayedfor TNF expression as described in U.S. Pat. No. 4,677,063, oressentially as described above, using the L929 assay. Of 10 clonestested, 5 exhibited between about 13 and slightly more than 70 units ofTNF/ml after 2 days of growing the transfected PA317 cells.

To show that PA317 pLTNFL6 transfected cells could produce infectiousvirions, transfected cells were seeded at a density of 3×10⁵ cells/100ml dish in Dulbecco's Modified Eagles Media supplemented with 10% fetalcalf serum and incubated for 16 hours. Subsequently, virus was harvestedby removing the media and subjecting it to centrifugation at 3,000×g for5 minutes to remove any cellular debris, as well as intact cells. Theresulting media supernatant containing virions was used to infectrecipient cell cultures.

Various cell types were infected and included NIH 3T3 cells, human tumorinfiltrating lymphocytes (TIL cells), or human melanoma cells. NIH 3T3cells are well known to those skilled in the art and readily accessible.Human TIL and melanoma cells were obtained as described by Rosenberg, S.A. et al., 1986, Science, 233:1318; Topalian, S. et al., 1988, J. Clin.Oncol., 6:839; Belldegrun, A. et al., 1988, Cancer Res., 48:206; orRosenberg, S. et al., 1988, New Eng. J. Med. Infection of the recipientcultures consisted of removing the cell culture media and replacing itwith virion containing supernatant culture media which was supplementedwith 4 μg/ml of polybrene to facilitate infection. Sixteen hours later,fresh media was added to the cultures, and the cultures metabolicallylabelled to permit determination of TNF by immunoprecipitation.

Metabolic labelling of the cells, as well as control cell lines known toproduce TNF, consisted of rinsing the cultures twice in phosphatebuffered saline without calcium or magnesium for the following celltypes: TNF6-8, NIH 3T3, and PA317 and incubating the cells incysteine-minus minimal essential media at 37° C. Melanoma cells wereincubated in Roswell Park Memorial Institute cysteine-minus media.Regardless of the type of media used, it was supplemented with 5%dialyzed fetal calf serum. The cells were then labelled with [35S]cysteine via aspirating off the media from the cell culture dishes, andreplacing it with media containing 100-300 μCi/ml of [35S] cysteine for3 hours at 37° C. Two ml of the radioactive media was used per 100 mmdish. Cultures were agitated during the labelling period after which themedia was aspirated and the cells lysed in 1 ml of lysis bufferconsisting of 20 mM Tris, pH 8.0, 200 mM LiCl, 1 mM EDTA, and 0.5%NP-40. Following the addition of the lysis buffer, the cell culturedishes were incubated for 5 minutes at 4° C., and the lysateimmunoprecipitated to determine the presence of TNF.

Immunoprecipitation consisted of precipitating TNF present in thelysates using rabbit anti-TNF sera which was raised against humanrecombinant TNF. Before the lysates were reacted with TNF antibody, theywere preabsorbed with 30λ of a 50% suspension of protein A SepharoseCL-4B beads for 1 hour at 4° C. with continual agitation. The tubes werecentrifuged to pellet the beads, and the supernatants transferred to newtubes to which was added 3.75λ of rabbit anti-TNF sera. The tubes wereincubated for 1 hour at 4° C. with agitation, and centrifuged for 15seconds at 14,000 rpm. The supernatants were removed and pipetted intonew tubes after which 30λ of protein A sepharose CL-4B beads was addedto the supernatants, followed by incubating the mixtures for 1 hour at4° C. with agitation. Next, the beads were pelleted and washed 3 timeswith lysis buffer, and 2 times with buffer B (20 mM Tris pH 8.0, 100 mMNaCl, 0.5% NP40). Washing consisted of pelleting the beads for 8 secondsat 7,000 rpm, discarding the supernatant, and adding 1 ml of theappropriate wash solution. Finally, after the last wash, the materialbound to the beads was subjected to electrophoresis by adding 30λ of gelelectrophoresis loading buffer consisting of 4% SDS, 15% glycerol, 6.25mM Tris pH 6.8, 0.1% bromophenol blue, and 1 mM DTT. Immediately afteradding the electrophoresis buffer, the samples were boiled for 5 minutesand subjected to electrophoresis on a 12% polyacrylamide gel. FIGS. 7and 8 show the results. FIG. 7 shows immunoprecipitation analysis ofcells transfected or infected with either the LTNFL6 plasmid or the hightiter TNF retrovirus derived from LTNFL6. Lane A, PA317 cellstransfected with the high titer retrovirus encoding both 26 kD and 17 kDTNF, LTNFL6; lane B, NIH 3T3 cells uninfected; and lane C, NIH 3T3 cellsinfected with the high titer TNF retrovirus, LTNFL6 clone 8 present inthe cell culture supernatant of LTNFL6 transfected PA317 cells.

FIG. 8 shows immunoprecipitation analysis of 35S-cysteine labelled humanmelanoma cells infected with the TNF retrovirus LTNFL6. Lane A, PA317cells transfected with LTNFL6; lanes B and C, uninfected and infected,respectively, NIH 3T3 cells with pLTNFL6 virus; and lanes D and E showuninfected and infected, respectively, human melanoma cells with thepLTNFL6 virus.

Confirmation of the integration of TNF encoding DNA sequences in humanTILs was obtained by polymerase chain reaction amplification of a 500base pair segment present in human TILs infected with pLTNFL6.

The reaction was conducted using standard PCR techniques and thefollowing oligonucleotide primers:

CP484: 5'-TNF primer

5'-GCT GGA GAA GGG TGA CCG AC-3'

CP487: 3'-MoMLV primer

5'-CTA TAG GCT TCA GCT GGT GA-3'

FIG. 9 shows the results. Lane A presents uninfected TILs, while lanes Band C show infected TILs. Lane D is a positive control and presentsPA317 cells transfected with pLTNFL6. The amplification of 500 base pairTNF sequence establishes that the TILs cells have been infected with theTNF retrovirus.

Additionally, immunoprecipitation experiments performed essentially asdescribed above showed that the TIL cells were secreting TNF.

pLIL-2L6

The vector pLIL-2L6 was generated using essentially the same materialsand methods described above that were employed to generate pLNL-MDR orpLNL.TNF with the following exceptions.

Firstly, the sequence that encodes IL-2 is present on a Pst I fragmentthat is described in detail in U.S. Pat. No. 4,518,584, inventor Mark etal.

Secondly, the oligonucleotide used to probe transformed bacteriacontaining the IL-2 sequence has the following sequence:

KD12: 5'-GAGTTGCATCCTGTACATTGTGGCAGGAGT-3'

The hybridization conditions were essentially those described that wereused to identify clones containing MDR or TNF sequences, except that theTm was 69° C. Screening for IL-2 clones revealed the presence of 8colonies by autoradiography that when restricted with Eco RI and Stu Iin a double digestion revealed clones that had the proper orientation ofthe IL-2 encoding sequence. Eco RI and Stu I digestion was carried outin a solution consisting of 31.5λ of 10×TA, 2.5λ of RNase A (10 mg/ml),252λ of glass distilled water, 13λ of Eco RI (20 units/λ), and 13λ ofStu I (8 units/λ).

EXAMPLE 6 Applications of pLMDRL6

The effectiveness of chemotherapeutic treatment of cancer is partlylimited by the dose that can be administered to a patient withoutuntoward side effects. These side effects include the elimination ofnormal lymphoid cells necessary for the immune responsiveness of thepatient. Thus, an important application of the retrovirus, pLMDRL6, isits capacity to confirm drug resistance on normal lymphoid cells,thereby permitting greater doses of a particular chemotherapeutic to beadministered.

The procedures and advantages of conferring drug resistance on lymphoidcells can be demonstrated by infecting tumor infiltrating lymphocytes,or TILs, with pLMDRL6 in vitro, and returning these cells to animmunologically receptive host animal experiencing achemotherapeutically sensitive tumor load. The procedures for obtainingTILs, as well as the procedures for infecting TILs with MDR amphotropicvirus are presented above. When the infected TIL cells are returned tothe animal an effective number of the cells adhere to, or infiltrate thetumor mass. Subsequently, the animal is administered one or morechemotherapeutics that are effective in treating the tumor load. Becausethe transfected TIL cells are resistant to concentrations ofchemotherapeutics that are toxic to the tumor, the physician maygradually increase the dose of chemotherapeutics that are effectiveagainst the tumor beyond the dose that could be given without killingthe TIL cells. In this way, a patients natural anti-tumor defenses arespared, and, indeed, augmented by exposing the tumor to elevated dosesof chemotherapeutics.

EXAMPLE 7 Two Gene Retroviral DNA Constructs

Retroviruses capable of expressing both the neomycin gene and TNF wereconstructed. The construction strategy for producing the two generetroviral vectors is shown in FIG. 10. The vectors pLNSX, pLXSN andpLNCX all carry the neomycin gene sequences. Further, these vectors aredescribed by Miller and Rosman, 1989, Biotechniques, 7(9):980.

The TNF construct contained the nucleotide sequence that encodes theγ-interferon signal peptide that is present in the vector pUC.FVXMΔHIIITNF γ sig. The procedure consisted of digesting 150 μg of pGEMTNF14 and150 μg of pUC.FVXMΔHIII TNF γ sig with 600 units Pst I (New EnglandBiolabs). The digestion was carried out in 1500 μl of restriction enzymebuffer consisting of 33 mM Tris-acetate, pH 7.9, 66 mM potassiumacetate, 10 mM magnesium acetate, 0.5 mM dithiothreitol, at 37° C. for60 minutes. The digests were phenol extracted, ethanol precipitated, andfractionated on a 1% Agarose gel in Tris-acetate electrophoresis buffer(40 mM tris, 1 mM EDTA, 5 mM sodium acetate, pH 7.5). The 1070 bpwild-type TNF fragment and the 902 TNF γ sig fragments, respectively,were isolated by glass beads.

Each Pst I fragment was made blunt by treatment with T4 DNA polymeraseFour μg of each fragment was treated with 6.3 units of T4 DNA polymerase(New England Biolabs) in a volume of 125 μl consisting 33 mMTris-acetate, pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate,0.5 mM dithiothreitol and 0.10 mM each of dATP, dCTP, dTTP, and dGTP, at37° C., for 10 minutes, then at 68° C., for 5 minutes. Each blunt-endfragment was purified by glass bead isolation.

Four μg of each vector was digested with an appropriate enzyme thatgenerated blunt ends, in a volume of 100 μl (33 mM Tris-acetate, pH 7.9,66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM dithiothreitol)as follows: pLNCX with 10 units Hpa I (NEB), pLNSX with 16 units STU I(NEB), pLXSN with 10 units HpaI (NEB), 37° C., 60 minutes.

Each digested vector was ligated to each blunt-ended TNF fragments in a25 μl ligation mixture that contained 16 μg/ml of vector and 16 μg/ml ofTNF fragment, 33 mM Tris-acetate, pH 7.9, 66 mM potassium acetate, 10 mMmagnesium acetate, 0.5 mM dithiothreitol, 4 mM ATP, 16,000 units/ml, T4DNA ligase (NEB). Ligations were incubated at 16° C., for 16 hours.Ligated DNA was transformed into competent DH5α cells (Bethesda ResearchLabs) following manufacturer's instructions. Resultant colonies werescreened by standard methods using TNF oligonucleotide primer CP365(5'-TCAGCTCCACGCCATTGG-3') as a probe. Orientation was confirmed byrestriction endonuclease analyses.

One of the plasmids carrying the TNF gene, pLTNFSN, was transfected intoPA317 cells, and transfected clones isolated. Virus was rescued from oneof the clones denoted LTNFSN-22. It was characterized, using standardtechniques, as having a titer of 1.6×10⁶ neo^(r) cfu/ml and produced 62units of TNF/ml.

Two gene viruses that encodes both neomycin resistance and IL-2 wereconstructed using the above described vectors, pLNSX, pLXSN and pLNCX.The procedures were similar with the following exceptions: a Pst Ifragment that encodes IL-2 was blunt ended and inserted into thevectors. The IL-2 fragment is described in U.S. Pat. No. 4,518,584,inventor Mark et al. IL-2 colonies were screened using theoligonucleotide KD12.

One of the plasmids carrying the IL-2 gene pLIL2SN was transfected intoPA317 cells, and transfected clones isolated. Virus was rescued from twoclones and denoted pLIL2SN-16, and pLIL2SN-20. They exhibited thefollowing titers, respectively, 2.0×10⁵ and 2.2×10⁵ neo^(r) cfu/ml andeach produced about 8 units/ml of IL-2.

Finally, a two gene construct was made using the TNF mutein, TNFΔ(1→12).Briefly, the Pst I fragment from pFVXMΔHIII TNFΔ12 was made blunt endedand cloned into the Hpa I site of pLXSN to generate pLTNFΔ12SN.

EXAMPLE 8 Synergistic Effect of TNF and Neomycin

A surprising property of cells infected with virions that encode TNF andneomycin resistance is their increased sensitivity to selection with theantibiotic G418, if G418 is added immediately after infection. This isshown by infecting cells with the virion pLTNFSN-22 and subjecting thecells to immediate selection in G418. A suitable target cell forinfection is PA317, and a suitable concentration of G418 is 400 ug/ml.

Using these conditions, about 60 percent of the cells that carrypLTNFSN-22 are killed in the presence of G418 at the end of 24 hours inthe selection media. In contrast, less than 10 percent of cells infectedwith the same virus, but lacking the TNF sequences are killed.

Without intending to be held to any particular theory, it is thoughtthat the sensitivity of cells expressing TNF in the presence of G418selection occurs because the infected cells have insufficient time toexpress the neomycin resistant phenotype. This, in turn, permits G418 toinhibit the synthesis of a protein that normally renders the cellsresistant to TNF killing. Thus, in the presence of G418 the cells arekilled by TNF.

Support for this hypothesis is borne out if G418 selection is notapplied to cells infected with pLTNFSN-22 until about 48 hours afterinfection, little cell death occurs during the subsequent 24 hours.

It will be appreciated by those skilled in the art that if cells thatharbor TNF virions are to be successfully isolated using G418, or othersimilar antibiotic selection, that there must be sufficient expressiontime for the neomycin gene to confer G418 resistance on the infectedcells.

EXAMPLE 9 Cytotoxic Properties of TNF Muteins

The following TNF deletion muteins of TNF, produced using theoligonucleotides described above, were tested for cytotoxic activity:TNFΔ(1→12), TNFΔ(1+12), and TNFΔ(1+13), that is, deletion of the first12 amino acids, of amino acids 1 and 12, and of amino acids 1 and 13,respectively. NIH 3T3 cells were co-transfected with β-actin-neo andwith one the series of pUC.FVXMΔHIII TNF plasmids and then selected in400 μg/ml G418. Cytotoxic activity was measured by seeding onto 100 mltissue culture dishes serial dilutions of the G418 resistantheterogeneous populations. After about 20-50 colonies of cells wereapparent, the media was aspirated off, and the colonies were overlaidwith about 4×10⁶ L929 cells in Dulbecco's Modified Eagles Mediumsupplemented with 10% fetal calf serum. Thirty minutes after plating thecells, the media was aspirated, and the cells overlaid with a solutionconsisting of 10 ml Dulbecco's Modified Eagles Medium, 0.9% Noble agar(Difco), and 10% fetal calf serum. This composition was kept molten at45° C. prior to overlay, cooled and overlaid onto the cells. Next, theagar was hardened at room temperature for 30 minutes, and the platesincubated at 37° C. for 48 hours, after which the agar was removed, 10ml phosphate buffered saline added, folllowed by 1 ml of a solutioncontaining 12% glutaraldehyde, 1% methylene blue. The latter solutionfixes and stains the cells which permits cell colonies and killing zonesto be readily visualized. Finally, the plates were incubated at roomtemperature for 60 minutes, and rinsed in water and air dried.

Cytotoxic activity was scored as killing zones surrounding thetransfected colonies. Two types of killing zones were observed; diffuseand non-diffuse zones. Non-diffuse killing indicates that the TNF muteinremains membrane bound and does not diffuse away from the transfectedcolonies to cause L929 death. In contrast, diffuse killing indicatesthat the TNF mutein is capable of diffusing away from the cell andcausing killing over a wide area.

Table I summarizes the results. In addition to the muteins that weretested for cytotoxic activity, a vector control, wild-type TNF, and TNFγ-sig were also tested for comparative purposes. It is apparent from thetable that only the vector control, and TNF Δ(1+13) do not exhibitcytotoxicity by the L929 overlay assay. Further, wild-type TNF and theTNF γ-sig have cytotoxic activity, but the activity exhibited a diffusepattern about the transfected colonies thus showing that wild-type TNFand TNF γ-sig are secreted from the transfected colonies. In contrast,TNF Δ(1→12), and TNF Δ(1+12) exhibit a non-diffuse killing pattern. Thisshows that these molecules exert their killing by being cell surfacebound and coming into direct contact with L929 cells.

                  TABLE I                                                         ______________________________________                                                   TNF Bioassay on  Bioassay by L929                                  Cell Population                                                                          Population Supernatants                                                                        Overlay                                           ______________________________________                                        Vector Control                                                                           <5               -                                                 TNF W.T.   134              +                                                 TNF Δ (1 → 12)                                                              <5               .sup. +.sup.b                                     TNF Δ (1 + 12)                                                                     <5               .sup. +.sup.b                                     TNF Δ (1 + 13)                                                                     <5               -                                                 TNF γ.sig                                                                          3125             +                                                 ______________________________________                                         Notes:                                                                        a. Positive results scored as diffuse killing zones surrounding target        colonies.                                                                     b. No diffuse killing zones about colonies but L929 cell killing observed     directly on cell colonies (cell to cell contact).                        

A second experiment was done to assess the cytotoxic activities of theinstant muteins, and particularly to confirm the cell surface locationof TNF Δ(1→12) and TNF Δ(1+12). The experiment consisted of two parts.First, cells harboring the various TNF muteins were cell surfaceradioiodinated using techniques well known in the art. The resultsrevealed that, surprisingly, both muteins are bound to the cell surface.Secondly, transfected cells expressing each of the TNF muteins weregrown in culture media, and labelled with ³⁵ S-cysteine after which themedia was harvested and immunoprecipitated with anti-TNF polyclonalantibodies. Such antibodies and immunoprecipitation methods are known inthe art. The immunoprecipitates were run on 12% polyacrylamid/SDS gels,and it was observed that for cells transfected with TNF Δ(1+12) and TNFΔ(1+13) there was a molecule about 17 kD that was immunoprecipitated.Nothing was detected in the TNF Δ(1→12) derived media.

The above results, when considered together, establish the following:TNF Δ(1→12), is membrane bound and not secreted into the cell culturemedia, and the membrane bound form is cytotoxic. TNF Δ(1+12) is alsomembrane bound and the membrane bound form is cytotoxic. Surprising,there is also secreted by the transfected cells a molecule about 17 kD.Finally, TNF Δ(1+13) appears to be membrane bound, but the membranebound form is not cytotoxic. This construct also yields a secretedmolecule of about 17 kD.

An experiment was conducted to determine if the soluble molecules havingmolecular weights about 17 kD secreted by cells transfected with TNFΔ(1+12) and TNF Δ(1+13) have cytotoxic activity. This experiment wasdone by growing the appropriately transformed 3T3 cells, and separatingthe cells and media containing the secreted molecules. The media wasassayed in L929 cells as previously described, and shown not to havecytotoxic activity. Thus, the secreted 17 kD molecules are, unlikewild-type 17 kD TNF, not cytotoxic.

FIG. 12 shows a restriction map of the cDNA sequence that encodes 26 kDTNF and the regions of the molecule that were deleted to produce thevarious muteins.

EXAMPLE 10 Therapeutic Applications for Two Gene Retrovirions thatEncode Neomycin Resistance and IL-2 or TNF

The virion pLIL2SN-16, described above, is utilized in tumor therapy byinfecting isolated human TIL cells using standard techniques. Theprocedures are described by Rosenberg, S. A. et al., 1986Science, 233:1318; Topalian, S. et al., 1988, J. Clin. Oncol., 6: 839; Belldegrun, Aet al., 1988, Cancer Res., 48: 206; or Rosenberg, S. et al., 1988, NewEng. J. Med. Infection of the recipient cultures consists of removingthe cell culture media and replacing it with virion containingsupernatant culture media which is supplemented with 4 μg/ml ofpolybrene to facilitate infection.

The TIL cells are washed to remove debris, polybrene, etc., and in otherways made suitable for infusion into a cancer patient. The dose ofinfected TIL cells may vary, with the optimal dose being empiricaldeterminable by the attending physician. It is anticipated that morethan one infusion of infected TIL cells will be desirable forsatisfactory treatment. Following the initial treatment and thereafter,the patients tumor mass is monitored and significant shrinkage would beapparent at the end of 4 weeks.

Using essentially the same procedures, TIL cells may be infected withretrovirions that encode the TNF mutein TNF Δ(1→12) using retrovirionsgenerated by transfection of pLTNF Δ12SN into an appropriate host cell,and isolating virions secreted into the cell culture media.

The following materials have been deposited at the American Type CultureCollection, Rockville, Md., USA (ATCC) under the terms of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure and Regulations thereunder(Budapest Treaty) and are thus maintained and made available accordingto the terms of the Budapest Treaty. Availability of such stains is notto be construed as a license to practice the invention in contraventionof the rights granted under the authority of any government inaccordance with its patent laws.

The following materials have been deposited with the ATCC and have beenassigned the indicated ATCC deposit numbers. They have also beendeposited with the Master Culture Collection (CMCC) of CetusCorporation, Emeryville, Calif., USA, the assignee of the presentapplication, and assigned the indicated CMCC deposit numbers:

    __________________________________________________________________________                     CTCC CMCC ATCC  Date of                                                       Deposit                                                                            Deposit                                                                            Deposit                                                                             ATCC                                         Plasmids         No.  No.  No.   Deposit                                      __________________________________________________________________________    pB11(pE4)             2138 39894 15 October 1984                              pAW711                2162 39918 8 November 1984                              pFVXM                 2701 67103 24 April 1986                                pLTNFL6               3684 68118 12 October 1989                              pLIL-2L6              3685 68119 13 October 1989                              pLMDRL6               3687 68120 13 October 1989                              p U.C. FVXM                                                                   Δ HIII TNF γ sig                                                                        3688 68121 13 October 1989                              pLTNFSN               3759                                                    pLTNF γ sig SN  3758                                                    pLNSTNF γ sig   3756                                                    pLNSTNF               3757                                                    pLNCTNF γ sig   3760                                                    pLNCTNF               3761                                                    pLIL2SN                                                                       Packaging Cell Line        CRL9078                                            PA317                                                                         Transfected NIH 3T3 Cells Lines*                                              TNF Δ (1 + 13) [TNFΔY]                                                             10729                                                        TNF γ sig  10726     CRL10333                                           TNF6 [wild-type TNF]                                                                           10730                                                        TNF Δ (1 → 12) [TNFΔ12]                                                     10728     CRL10334                                           TNF Δ (1 + 12) [TNFΔW]                                                             10727                                                        __________________________________________________________________________     *Cells were transfected with pUCFVXM Δ HIII containing the              appropriate TNF construct.                                               

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The deposit ofmaterials herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is thisdeposit to be construed as limiting the scope of the claims to thespecific illustrations which materials deposited represent.

We claim:
 1. DNA encoding the γ-interferon signal peptide of FIG. 1directly linked to DNA encoding mature TNF, wherein said signal peptidehas the property of augmenting the secretion of said mature TNF frommammalian cells.
 2. A vector capable of expressing DNA in mammaliancells, said vector comprising DNA encoding the γ-interferon signalpeptide of FIG. 1 directly linked to DNA encoding mature TNF, whereinsaid signal peptide has the property of augmenting the secretion of saidmature TNF from mammalian cells.
 3. A mammalian cell comprising thevector of claim
 2. 4. A mammalian cell comprising the DNA of claim 1.